1. Field of the Invention
The present invention relates to modified sarcosine oxidases, genes and recombinant DNAs thereof, and methods for preparing the same.
2. Background Art
Sarcosine oxidases are enzymes with a catalytic activity to hydrolyze sarcosines to produce glycine and formaldehyde, which can be used to measure the amount of creatinine in human serum or urine, or can be utilized as diagnostic agents for various diseases such as renal disease.
Sarcosine oxidases are previously known to be produced by bacterial strains such as Corynebacterium genus (see J. Biochem., 89,599 (1981)), Bacillus genus (see JP Patent Publication (Unexamined Application) No. 54-52789), Cylindrocarbons genus (see JP Patent Publication (Unexamined Application) No. 56-92790), Pseudomonas genus (see JP Patent Publication (Unexamined Application) No. 60-43379), Arthrobacters genus (see JP Patent Publication (Unexamined Application) No. 2-265478). However, in typical creatine quantitative reactions in slightly alkaline range (pH 7.5-8.0), bilirubin affects the measurement value, thus causing a difficulty upon measuring. Until now, genetically modified enzymes having optimal pH in the slightly acidic range, which are produced by modifying sarcosine oxidase genes from Bacillus genus, aiming for measuring in the slightly acidic range where effects of bilirubin are less, are also known (see JP Patent Publication (Unexamined Application) No. 2000-175685). However, these modified enzymes were not practical with respect to instability in the slightly acidic range.
As described above, when enzymatically quantifying creatinine or creatine, the reliability of the measurements are reduced if substances such as bilirubin affect them, since the amounts of the two substances in serum or blood are extremely small. It is also known that when measurements are performed in the slightly acidic range where the effects of bilirubin etc. are small, decrease in stability occurs. To solve these problems with improvements other than of enzymatic nature, the amount of sarcosine oxidase in the active formulation had to be increased than it is normally used; buffers had to be selected appropriately; or additives had to be added. However, improvements by such means could increase the cost of measurement reagents and therefore were not practical.
The object of the present invention is to provide sarcosine oxidases which have optimal pH and show high activity in the slightly acidic range, which also have improved stability.